The present invention generally relates to the field of bio-technology, genetic research, and DNA diagnostics, and it more specifically relates to an automated high speed thermal cycling system for carrying out temperature controlled processes, including but not limited to polymerase chain reactions for identifying and amplifying specific elements of a genetic sequence in a sample of material.
A rapidly growing technique being employed by many molecular biology laboratories is the amplification of DNA sequences through polymerase chain reaction (PCR), a technique which utilizes thermal cycling systems. The PCR process was introduced in the 1980's and has revolutionized genetics-related research. PCR replicates a small amount of DNA in a series of heating and cooling cycles, and has been used in diverse research applications, including molecular biology DNA sequencing, cloning, research into mutagenesis and gene synthesis using published DNA sequences.
Generally, thermal cyclers allow the PCR process to proceed automatically by subjecting the reagents-DNA nucleotides and a heat-tolerant polymerase, among others, to a user-specified heating and cooling sequence. Research analyses usually require many copies of a particular targeted DNA segment. PCR is a relatively quick and highly efficient way to copy or amplify DNA molecules in a test tube without needing cell cultures. PCR allows researchers to amplify any DNA sequence regardless of its origin (i.e., virus, bacteria, plant, or any human cell) hundreds of millions of times in a matter of hours.
PCR is especially valuable because the reaction can amplify extremely small amounts of starting material. Thus, it has had a major impact on clinical medicine, genetic research and diagnosis, and evolutionary biology as well as forensic science, and has allowed a spectrum of advances ranging from the identification of novel genes and pathogens to the quantization of characterized nucleotide sequences.
The PCR process is based on a special polymerase enzyme (a protein acting as a catalyst) that catalyzes the synthesis of a new strand of DNA complementary to an existing target strand. The starting mixture contains the DNA sample of interest, the four building blocks of DNA (called DNA bases), and two DNA fragments (called primers) that flank the target sequence.
A single PCR cycle includes three steps: denaturation, primer binding or annealing, and DNA synthesis or extending. During denaturation, the starting mixture is first heated to about 94.degree. C. to 96.degree. C. for separating the double strands of DNA. After denaturation of the DNA, the mixture is cooled to about 55.degree. C. to allow the primers to bind to their complementary sequences on the separated strands. The primers define the ends of the DNA to be duplicated. Then, the mixture is heated to a temperature of about 72.degree. C., so that the DNA polymerase catalyzes the extension of the annealed primers on the template strand.
Repeated cycles of denaturation, primer annealing, and primer extension result in the exponential multiplication of the target DNA because each new double strand separates to become two new DNA templates for further synthesis. Some 20 cycles of the PCR can amplify the target DNA by a factor of a million in about one to two hours.
A typical instrument for automating PCR comprises a temperature-controlled sample block having a plurality of wells. A block may have, for example, 96 such wells in an 8.times.12 format. Each well receives a thin-walled reaction tube having a volume accommodating samples in the range of 10 to 100 .mu.l. Nucleic acids and reagents are placed within these tubes which are then placed into the wells, in the temperature-controlled sample block. The system is then cycled through a heating and cooling sequence for achieving the desired DNA amplification.
It is important that thermal cyclers reach appropriate temperatures quickly and provide a uniform temperature over all the samples, to ensure accurate and uniform results. This is difficult to achieve, and manufacturers of thermal cyclers have turned to different technologies for thermal cycling, i.e., cyclically heating the samples and then cooling them down. Most, but not all, use an electrically heated element to deliver heat to a metal plate that surrounds the sample tubes. The heating cycle of such an element is relatively long, and its temperature can sometimes exceed a predetermined upper limit.
For cooling, several approaches are used. Some models do not offer active control when it comes to cooling, they simply let excess heat escape into the ambient air. These are the least expensive instruments to manufacture, but they can have uniformity problems. Another method of cooling is that used by Perkin-Elmer, one of the largest manufacturers of thermal cyclers. This approach relies on vapor compression heat pumping, which is similar to a typical refrigeration unit. Other devices cool the samples using running water which is then discarded. However, these devices consume an extensive amount of energy, and are not energy efficient.
Another technology used in thermal cyclers is an electronic process called the Peltier effect. Depending on the direction of the electrical current in a Peltier unit, which includes two ceramic outer layers sandwiching an inner layer of semiconductor material, it can actively cool one surface while heating the other. This effect can cause a temperature differential between the surfaces. Reversing the flow of the current reverses the flow of heat, but the cooling capacity of these units is limited.
Conventional thermal cyclers suffer from drawbacks and inaccuracies, among which are the following: In most of these thermal cyclers heat transfer to the reagents contained in the tubes is slow, and the overall cycling time is relatively long, such as 2 hours for the entire amplification run. Furthermore, the biological reagents are subjected to substantial periods of time at intermediate non-ideal temperatures, allowing undesirable reactions to occur. Under these conditions, and depending on the primers used, there can be nonspecific priming, resulting in the amplification of undesired DNA. Research studies have shown that this undesired "background" can be decreased by increasing the rapidity and accuracy of the thermal cycling.
Therefore, it would be desirable to have a new thermal cycling system for effective use in various applications and temperature controlled processes, including but not limited to polymerase chain reactions. This new thermal cycling system should contain samples in the form of small volumes of liquid, typically between 10 and 50 .mu.l. It should automatically and simultaneously process a large number of samples.
The new thermal cycling system should maintain precise control of the temperature cycle. In particular the reagent temperatures must not exceed the highest reaction temperature (i.e., 96.degree. C.), otherwise, the PCR enzyme is rapidly destroyed. The new cycling system should allow a rapid and almost instantaneous change of temperatures between the three steps of each single PCR cycle: denaturing (about 94.degree. C.), annealing (about 55.degree. C.), and extending ( about 72.degree. C.), in order to minimize non-specific binding, i.e., incomplete identification, which may occur at intermediate temperatures.